Project Summary Growth and homeostasis of bones require calcium and inorganic phosphorus (Pi). With millions of Americans suffering from bone mineralization disorders, there exists a need for understanding processes governing hard tissue mineralization. Pi is implicated in not only forming hydroxyapatite, the main crystal giving bone its compressive strength, but also regulating osteoblast and chondrocyte differentiation and function. PiT-2 (gene: Slc20a2) is the major form of sodium-dependent Pi transporters in mineralized tissue. Our laboratory is one of the first to have identified its potent role in regulating skeleton development and mineralization. Knockout of the PiT-2 gene in mice resulted in tooth and bone mineralization abnormalities, as evidenced by reduced osteoblast numbers, bone density and volume, and impaired incisor development and amelogenesis. The focus of this research is to explore further the role of PiT-2 in cellular and extracellular processes governing skeletogenesis, dentinogenesis, and amelogenesis. Specifically, Aim 1 will confirm and extend our preliminary findings to determine whether PiT-2 is required for regulation of odontoblast and ameloblast numbers, differentiation, and/or mineralizing activity with deficiencies leading to impaired dentinogenesis and amelogenesis. A role of PiT-2 in dentin repair and tertiary dentin formation will also be studied using a molar injury mouse model. In Specific Aim 2, we will define the mechanisms of action of PiT-2 in mineralizing cells using osteoblasts as an example. A Pi transport and sensing/signaling function of PiT-2 will be dissected through engineered PiT-2 transport deficient mutants. We expect these studies to provide a better understanding of how Pi influences functions of hard tissue forming cells, serving as a basis to develop novel therapeutics for patients suffering from mineralization disorders, such as osteoporosis and osteopenia. This project will take place at the University of Washington with collaborations between the Departments of Bioengineering and Oral Health Sciences during DDS-PhD training of the PI. The PI will develop critical thinking skills, learn state-of-the-art cellular and molecular techniques, in vivo transgenic mouse models, and data analysis that will prepare him for independent research. Ultimately, a strong mentorship team, multidisciplinary collaborative effort, and resources at the University of Washington with support through this award will prepare the trainee for a future tenure-track faculty position to study biomineralization processes.